Distribution and clinical role of KIT gene mutations in melanoma according to subtype. A study on 492 Spanish patients

Background KIT mutations are primarily associated with acral and mucosal melanoma, and have been reported to show higher prevalence in chronic sun-damaged (CSD) than non-CSD melanomas. Objectives To investigate the prevalence of KIT mutations in melanoma according to subtype, and to determine the clinical role of such mutations. Material and Methods Here we present results from a study on a Spanish population of 492 melanomas, classified according to the latest World Health Organization (WHO) guidelines. We analyzed the mutational status of KIT and correlated with different clinical variables related to sun exposure and family history. Results KIT mutations were significantly more frequent in acral (3/36; 8.3%) and mucosal (4/8; 50%) melanomas than in non-acral cutaneous melanomas. No significant difference was observed in KIT mutational status between CSD and non-CSD melanomas. Conclusion Our results suggested that KIT mutations in melanoma tumors are unrelated to nevus proneness or chronic sun damage, but their presence is associated with aggressive melanomas showing ulceration, vascular invasiveness, and increased Breslow thickness. These findings were consistent with those reported by The Cancer Genome Atlas network.


Introduction
Activating mutations in the gene encoding KIT receptor (KIT) lead to persistent upregulation of MAPK and PI3K signaling cascades without a ligand (1,2).Several studies have shown mutations in the gene mainly in cutaneous acral and mucosal melanomas in 15-30% of patients.(3)(4)(5).As per a divergent pathway hypothesis, melanomas can arise via nevogenic pathway where host factors are critical, and patients present melanocytic instability.In such patients, melanomas are generally located at unexposed or intermittently sun exposed areas of skin without chronic sun damage (CSD).Such lesions usually carry BRAF mutations.Alternately, the CSD pathway through cumulative sun exposure leads to melanomas at usually exposed body sites with a substantial degree of solar elastosis that are characterized by a relatively increased proportion of NRAS and KIT mutations (6)(7)(8)(9)(10).Only a few studies have investigated the prevalence of KIT mutations in non-acral cutaneous melanoma (11) as most studies have been focused on acral and mucosal melanomas (12,13) (Table 1); In this study, we performed a retrospective investigation on the mutational status of KIT gene in primary tumors from a large number of Spanish melanoma patients that included all melanoma subtypes.We determined the association between KIT mutations and various clinical variables including different tumor characteristics.Our data showed that the frequency of KIT mutations was low in non-acral melanoma and that, contrary to previously reported, prevalence was not different between CSD and non-CSD melanomas.Besides, our evidence suggests the role of KIT mutations as an aggressiveness marker.

Materials and methods
We designed a retrospective study using the melanoma database of the Instituto Valenciano de Oncología (IVO), which contains prospectively collected information from all melanoma patients treated at the institute, a tertiary referral oncology hospital in Spain.The data had been collected from January 2000, included clinical, pathological and epidemiological information assessed by two expert dermatologists at first visit (E.N. and C. R.) (14).The study had the approval of the Ethics Committee at IVO and informed consent from patients.

Sample recruitment and classification
Melanoma patients included in the study were diagnosed between 2000 and 2018 at IVO. Tumor samples were collected and stored in the Biobank.Melanoma diagnosis was pathologically confirmed by a single pathologist (V.T.).Patients were classified based on the presence/absence of KIT mutation.Additionally, patients were classified according to the latest WHO classification of melanomas (non-CSD, CSD, acral and mucosal) (15).In this classification, the difference between non-CSD and CSD was based on the degree of solar elastosis in the unaffected skin surrounding the melanoma.
Sanger sequencing was performed using 10pM of each primer and the ABI Prism BigDye Terminator Cycle Sequencing kit (Applied Biosystems®).The sequencing conditions were: 96°C-1 min; 25 cycles of 96°C-10 sec, 50°C-5 sec, 60°C-4 min; 4°C hold.Resulting sequencing products were purified via ethanol precipitation and sequenced according to established protocols in a Sanger sequencer (3031x Genetic Analyzer; Applied Biosystems®).

Statistical analysis
Categorical variables included clinical characteristics and mutational status for KIT gene.A Chi square test was applied to study differences among the groups, using a threshold of p<0.05 to define statistical significance.First analysis included all selected samples, and the second analysis was carried out by excluding acral and mucosal subtypes (Tables 2 and Supplementary Material).
Melanoma-specific and overall survival were calculated using the Kaplan-Meier method and differences between the curves were tested by log-rank test.The statistical analyses were performed using IBM Corp. Released 2011 (IBM SPSS Statistics for Macintosh, version 20.0.Armonk, NY: IBM Corp.).
KIT mutations were more frequent (p=0.017) in tumors at the sites without sunburns (13/193; 6.7%) than at the sites with either moderate or severe sunburns (5/281; 1.8%).Similarly, the frequency of mutations in melanomas at rarely exposed sites (8/83, 9.6%) was higher (p=0.014)than in tumors at usually or occasionally exposed sites (13/409; 3.2%).The difference in the mutation mutations frequency based on anatomical sites of primary tumors was also statistically significant (p<0.001).The frequency of the mutations in tumors with ulceration was 9.2% (12/130) and 2.5% (9/361) in tumors without ulceration (p=0.004).None of the nevus-associated melanomas had KIT mutation, and none of the melanomas harboring BRAF mutation carried a KIT mutation.No KIT mutations were detected in lentigo maligna melanoma (LMM) (Table 2).
We also analyzed the data after exclusion of patients with mucosal and acral melanomas (Supplementary Material).The frequency of KIT mutation in all other tumors was 3.1 % (14/448).We observed no statistically significant differences in mutation frequency in tumors from CSD sites (3/64; 4.7%) and non-CSD sites (11/384, 2.9%).The anatomically distribution of the KIT mutations was significantly different (p<0.001)(Supplementary Material).
There was no statistically significant association between KIT mutations and family history of melanoma or other cancer type in either entire set of patients or in patients without mucosal and acral melanomas.A trend was observed associating mutations in KIT gene with vascular invasion and thicker melanomas (Table 2 and Supplementary Material).
After a median follow-up of 72 months, 108 patients died, 70 of which were due to melanoma.5-year estimated melanoma-specific and overall survival of patients with KIT mutated melanomas was 77.6% and 73.3%, whereas those without mutations was 82% and 87.5%.Neither comparison showed statistically significant differences (Supplementary Material).

Discussion
In this study based on the prevalence of KIT mutations in melanoma, a large patient set confirmed the high occurrence of such mutations in acral and mucosal melanomas.However, in CSD and non-CSD melanomas, we did not observe the previously reported difference in KIT mutation prevalence.This was further corroborated with TCGA data, given that tumors mutated for KIT did not associate with signature 7, which is attributed to UV exposure.We also suggested that KIT mutations defined aggressiveness in melanoma, which is consistent with TCGA data.Cancer Genome Atlas Network, 2015), but differed in the most prevalent mutated subtype (Supplementary Material).However, it may be pointed out that the number of acral and mucosal melanoma which are generally associated with a high frequency of KIT mutations, was low in our dataset.
Our results differ from previous studies reporting a higher prevalence of KIT mutations in CSD melanomas (9,10).In fact, we found no significant difference in KIT mutation prevalence between CSD and non-CSD tumors.Clinically, melanomas developed at usually sun-exposed skin and with a past history of sunburns were not associated with KIT mutations.Interestingly, none of the LMM, the paradigmatic type of chronic sun exposure-associated melanoma, harbored KIT mutation.Thus, these findings strengthened the hypothesis that KIT mutation are acquired in a CSD-independent manner.Furthermore, we showed that most KIT mutated melanomas seemed not to follow a nevogenic development pathway either, given the lower number of nevi in melanoma patients with KIT mutated melanomas and that none of the nevus-associated melanomas presented KIT mutations.Hence, the development of KIT mutations would be independent of the common etiopathogenic pathways.Also, KIT mutations have previously been found to determine a worse melanoma survival (19).In our cohort, survival was worse in KIT-mutated melanomas but differences were not statistically significant, most likely due to the small number of KIT-mutated cases.However, our pathological results do provide further evidence that links KIT mutations and aggressive melanomas because of the association with the presence of ulceration, vascular invasion and increased Breslow thickness in our patients.This aggressiveness could be expected given the role of KIT receptor in the cell.KIT mutations lead to the constitutive activation of the receptor, which in turn triggers both MAPK/ERK and PI3K/AKT signaling cascades (9).As a result of their activation, processes such as cell growth and proliferation are enhanced and pro-apoptotic signaling is reduced (20).Moreover, the fact that mutations affect different domains of the protein both extracellular and intracellular makes it more challenging to develop targeted therapies (2).
Our findings were consistent with the information published by the TCGA network.We performed analyses with available sequencing data to check the association between KIT.Firstly, concerning the aggressive profile, KIT mutations were significantly overrepresented in the "keratin-high" RNA expression group, which was associated with the worst survival.Secondly, the low relation with sun exposure was supported by the fact that KIT-mutated melanomas showed a less prevalence of the UV damage-associated Signature 7 (Supplementary Material).
In conclusion, we showed here the so far largest study on KIT mutations in melanoma patients for a Spanish population.Our results supported the role of KIT mutations as an aggressiveness marker for melanoma patients and suggested that the development of the pathogenesis of KIT-mutated melanomas is independent of the common etiopathogenic pathways (nevus-proneness and chronic sun damage).

Supplementary Material
Refer to Web version on PubMed Central for supplementary material.

Table 2 Distribution of melanomas tested for KIT among clinical variables.
CSD Eur J Dermatol.Author manuscript; available in PMC 2023 September 06.Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts Millán-Esteban et al.Page 13 Eur J Dermatol.Author manuscript; available in PMC 2023 September 06.Europe PMC Funders Author Manuscripts Europe PMC Funders Author Manuscripts Millán-Esteban et al.Page 14